Most Butyrivibrio strains have been isolated from the gastrointestinal tract of animals and have been classified as Butyrivibrio fibrisolvens. A few strains isolated from human feces are designated as Butyrivibrio crossatus, the other species in this genus. Butyrivibrio fibrisolvens strains are anaerobic, curved rods that produce butyrate, but numerous studies have shown that these strains display considerable variations in phenotypic properties and heterogeneity in DNA relatedness. Although over 60 strains have been characterized in these respects, the cell wall structure of only a few strains has been studied. In this study, cell wall related properties of 12 strains representative of five DNA relatedness groups were examined. All strains were very sensitive to penicillin and other antibiotics that interfere with cell wall synthesis. Although an occasional resistant strain was found, most strains were sensitive to a variety of protein synthesis antibiotics that included aminoglycosides and tetracycline. In contrast, all strains were highly resistant to nalidixic acid. Peptidoglycans were isolated from seven B. fibrisolvens strains and Lachnospira multiparus. Compositional analyses indicated molar ratios of 0.7:2:2:1:0.8 for muramic acid, glucosamine, alanine, glutamic acid, and diaminopimelic acid, respectively, in all peptidoglycans, which also showed a low degree of cross-linking. A trichloroacetic acid extractable galactosamine-containing polysaccharide copurified with the Butyrivibrio peptidoglycans. Electron microscopy of thin sections showed all strains to possess a Gram-positive type of cell wall that was atypically thin (12–18 nm). Most strains also displayed external (surface) polysaccharide layers. Cytoplasmic inclusions and granules were evident in many strains and were composed of polysaccharides, on the basis of cell composition analyses. The findings that Butyrivibrio strains have overall similarities in cell wall properties, but differences in DNA relatedness, suggest that these organisms should be classified as several more species in the same genus or family.Key words: Butyrivibrio fibrisolvens, Butyrivibrio crossatus, cell wall, peptidoglycan, ruminal bacteria.